The gene therapeutic treatment by use of viral vectors is a promising treatment option for diseases that do not respond or do not sufficiently respond to conventional treatment. This approach is based on the introduction of therapeutic genes into the organism to be treated using viruses which have been modified to include the sequence of the respective gene in their genome. Viral vectors which have been used for gene therapy in gene therapeutic approaches are based on retroviruses, lentiviruses, adenoviruses and adeno-associated viruses.
Adeno-associated viruses (AAV) are promising candidates for use in clinical practice because they are considered as relatively safe. AAV vectors are capable of introducing a transgene into a tissue and expressing same stably and efficiently. At the same time, these vectors do not possess any known pathogenic mechanism [1]. The AAV vectors of serotype 2 (AAV2) are of particular importance for clinical use, and these vectors have been examined particularly well. After introduction by AAV vectors, the transgenes can be present within the transfected cell in different forms, for example as episomal, single-stranded or double-stranded DNA. Concatemeric forms of DNA were also found in transduced cells.
The genome of AAV2 is a linear, single-stranded DNA molecule of approximately 4700 nucleotides in length, and it comprises inverted terminal repeats (ITRs) at both ends. The genome further comprises two large open reading frames which are referred to as replication region (rep) and capsid region (cap). The replication region encodes proteins which are required in connection with virus replication. In contrast, the capsid region encodes the structural proteins VP1, VP2 and VP3 which make up the icosahedral capsid of the virus.
As most of the vectors which can be used in gene therapy and which are known in the state of the art, wild-type AAV vectors, e.g. the disclosed AAV2 vectors, do not possess sufficient specificity for a specific tissue but instead infect a wide variety of cell types. Upon systemic administration of these wild-type vectors, the target tissue is insufficiently transduced, and severe immune reactions have to be expected in the treated patient due to the undesired transduction of other tissues. Progress has been made with the development of viral vectors having an increased specificity for particular organs by using peptide ligands that are capable of guiding the vectors to specific organs [2-3]. It could be shown that specific peptide ligands provide for a “homing” to different organs such as the brain.
Reference [4] describes a method that allows for the screening of capsids of AAV2 with modified tropism in randomized peptide libraries. From these libraries, vectors can be isolated that specifically transduce a desired cell type in vitro. However, it has been found that capsids selected in this way are often unsuitable for being used in vivo since the required specificity is missing in an animal model [5].
From the clinical perspective, the brain is an extremely relevant organ, since it is the starting point of a variety of neurological diseases. Considerable efforts have been made to make this organ accessible for gene therapeutic interventions [6-12]. However, the blood brain barrier, which is composed of endothelial cells, pericytes and astrocytes and which seals the brain off from circulating particles, toxins and signal compounds, represents a barrier that cannot be passed by normal vector systems. Since suitable vector systems that transduce the brain with sufficient efficiency after intravenous application are not available so far, present gene therapy vectors are normally injected directly into the brain [13] which is associated with a higher risk for the patient. The possibility of making the blood brain barrier permeable for a short time, e.g. by ultrasound [14] or by chemical agents [7] is also regarded as very risky.
Accordingly, there is a high demand for agents, which are capable of modulating the tropism of viral vectors, thereby providing for a sufficient cell or tissue specificity which renders possible a targeted transport of a viral vector to tissues of the brain or spinal cord. Such vectors can provide for the specific expression of therapeutic genes in these tissues, thereby effectively treating diseases and/or functional conditions of the brain and the spinal cord.
The present invention provides viral vectors for the targeted gene transfer into the brain and spinal cord. The viral vectors of the present invention express on their capsid surface so far unknown amino acid sequences that are specifically recognized in vivo by receptors on the neurons of the brain and the endothelial cells of the blood vessels of the brain and spinal cord. In this way, the viral vectors of the present invention, after systemic administration to a subject, specifically transduce the tissues in the brain and spinal cord.
The viral vectors of the present invention further enable a strong and long persisting expression of a transgene in the neurons of the brain and in the endothelial cells of the blood vessels of the brain and spinal cord. Therefore, the vectors are particularly suitable for the gene therapeutic treatment of defined diseases or conditions of the brain and spinal cord. It has been furthermore found that the AAV vectors produce only a slight immune reaction after transfection in the host, and they are therefore particularly suitable for gene therapy.